Understanding the mechanisms by which cells produce, transmit and sense forces constitutes a next frontier in cellular and developmental biology. Major advances have improved our understanding of the biophysics of chemomechanical force production by motor proteins in vitro. Nevertheless, how such proteins collaborate with the dynamic cytoskeleton in vivo to produce forces that drive cell shape changes for morphogenesis, cytokinesis and cell locomotion is unclear. Moreover, how such forces cause changes in gene expression and signaling also remain challenging unanswered research questions. We propose to construct and characterize a family of genetically encoded myosin force sensors designed to quantitatively map forces in vivo with subcellular resolution. Our approach is based on successful force sensors in vinculin and -actinin. Our focus is on the forces produced by ensembles of nonmuscle myosin II in Drosophila, which are essential for cytokinesis and morphogenesis. Specific Aim 1: We will construct genetically encoded Tension Sensing Modules (TSMs) based on Fluorescence Resonance Energy Transfer (FRET) pairs separated by protein springs. We will systematically test a) different FRET pairs; b) different protein springs; and c) different locations in the myosin motors. These myosin force sensors will be expressed in both fly cell lines and transgenic flies. Specific Aim 2: We will characterize the myosin force sensors (MFSs) in fly cell lines. We will evaluate their ability a) to localize with endogenous myosin; b) to sense tension, using force traction microscopy on flexible substrates; and c) to respond to acute changes in tension induced by drugs or laser surgery. Appropriate control constructs will verify that changes in FRET are due to changes in force across the sensor. Specific Aim 3: We will characterize the myosin force sensors in transgenic flies. We will evaluate their ability a) to rescue myosin function in specimens depleted of endogenous myosin; b) to sense tension in dorsal closure (myosin II) and c) to report acute changes in tension induced by drugs or laser surgery. Specific Aim 4: We will calibrate the myosin force sensors that work in cultured cells and in fly specimens using single-molecule and/or solution strategies in vitro. Our experiments will deliver optimized myosin force sensors and the first measurements of the forces produced by ensembles of myosin in vivo. They will provide new information about the biophysics of cell shape changes in cytokinesis and morphogenesis. Because myosins play key roles in a variety of processes fundamental to human homeostasis and development, we expect these force sensors to have broad impact. Moreover, they will contribute to the resolution of several existing controversies regarding the role of myosins in biology. PUBLIC HEALTH RELEVANCE: Living cells are the fundamental building blocks of all life and respond to and influence their environments in a variety of ways. Here, we focus on how cells generate and respond to mechanical forces by proposing to design and implement genetically encoded tension sensing modules that will allow us monitor when and where cells generate forces as they grow, divide, crawl and change shape. This multidisciplinary research will impact human health because it addresses the basic biological problem of how myosin motor proteins produce forces for development and homeostasis.